中国普通外科杂志 ›› 2021, Vol. 30 ›› Issue (3): 294-304.doi: 10.7659/j.issn.1005-6947.2021.03.007

• 基础研究 • 上一篇    下一篇

骨髓间充质干细胞对急性重症胰腺炎大鼠的炎症抑制作用及对TLR4/NF-κBp65通路的关系

秦亚飞1,霍星光2,李秋霞1,徐玉生3   

  1. (1. 河南省周口骨科医院  重症医学科,河南 周口 466000;2. 河南省周口市卫生计生监督局  公共卫生监督二科,河南 周口466000;3. 郑州大学第一附属医院  骨科,河南 郑州 450000)
  • 收稿日期:2020-09-23 修回日期:2021-02-17 出版日期:2021-03-25 发布日期:2021-04-06
  • 通讯作者: 秦亚飞, Email: 13183228866@126.com
  • 作者简介:秦亚飞,河南省周口骨科医院副主任医师,主要从事急、危、重症患者救治及基础医学方面的研究。
  • 基金资助:
    河南省高等学校重点科研资助项目(18A320010)。

Anti-inflammatory effect of bone marrow mesenchymal stem cells in rats with severe acute pancreatitis and its association with TLR4/NF-κBp65 pathway

QIN Yafei1, HUO Xingguang2, LI Qiuxia1, XU Yusheng3   

  1. (1. Department of Critical Care Medicine, Zhoukou Orthopedic Hospital, Zhoukou, Henan 466000, China; 2. the Second Public Health Supervision Division of Zhoukou Health and Family Planning Supervision Bureau, Zhoukou, Henan 466000, China; 3. Department of Orthopedics, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China)
  • Received:2020-09-23 Revised:2021-02-17 Online:2021-03-25 Published:2021-04-06

摘要: 背景与目的:急性重症胰腺炎(SAP)为临床常见急危重症之一,炎症反应在其发生发展中发挥重要作用。目前认为骨髓间充质干细胞(MSC)移植具有修复SAP胰腺组织损伤潜能,但具体功能尚不清楚。本研究在SAP大鼠模型上探讨骨髓MSC对SAP炎症的抑制作用及机制。
方法:分离、培养、鉴定SD骨髓MSC,并制成1.0×106/mL的悬浮液;以3.5%牛磺脱氧胆酸钠逆行胰胆管注射建立SD大鼠SAP模型,取造模成功36只大鼠随机均分为4组,分别于造模后12 h后腹腔注射生理盐水(模型组)、腹腔注射MSC悬浮液(腹腔注射组)、尾静脉注射MSC悬浮液(尾静脉注射组)、腹腔联合尾静脉注射MSC悬浮液(联合注射组),注射剂量10 mL/kg;另取9只大鼠行假手术(假手术组),并在术后12 h腹腔注射等量生理盐水。治疗后24 h,处死各组大鼠获取血液与胰腺组织标本。用HE染色法观察胰腺组织病理形态学,进行病理评分;采用碘比色法检测大鼠血清淀粉酶(AMS)水平,甲基试卤灵底物法检测血清脂肪酶(LPS)水平;ELISA法检测胰腺组织IL-6、IL-1β、TNF-α含量;羟胺法检测过氧化物歧化酶(SOD)活性,硫代巴比妥酸法检测丙二醛(MDA)浓度;RT-PCR检测Toll样受体4(TLR4)、髓样分化因子88(MyD88)、NF-κBp65的mRNA相对表达量;Western blot法检测TLR4、MyD88、NF-κBp65、p-NF-κBp65蛋白表达量。
结果:骨髓MSC呈梭形、漩涡样生长;低表达CD45(1.25%),高表达CD90(99.89%);细胞活力良好。与假手术组比较,其余各组胰腺组织均有不同程度的病理改变,血清AMS与LPS水平、胰腺组织IL-6、IL-1β、TNF-α、MDA含量升高,而SOD活性降低,以及TLR4、MyD88 mRNA与蛋白相对表达量、p-NF-κBp65/NF-κBp65升高(均P<0.05)。但以上指标的变化程度在模型组、腹腔注射组、尾静脉注射组、联合注射组依次明显降低,差异均有统计学意义(均P<0.05)。
结论:骨髓MSC移植可抑制SAP大鼠炎症反应,减轻氧化应激反应,修复胰腺组织损伤,作用机制可能与调控TLR4/NF-κBp65通路有关。

关键词: 胰腺炎, 急性坏死性;间质干细胞移植;炎症;Toll样受体4;大鼠

Abstract: Background and Aims: Severe acute pancreatitis (SAP) is one of the most frequently encountered serious acute illnesses in clinical practice. Inflammation plays an important role in its occurrence and development. At present, bone marrow mesenchymal stem cell (MSC) transplantation is considered having the potential to repair the pancreatic tissue injury caused by SAP, but the specific function is unclear. Therefore, this study was conducted to explore the anti-inflammatory effect of bone marrow MSC and its mechanism in SAP using SAP rat models. 
Methods: The bone marrow MSCs were isolated from the SD rats, and then were cultured and identified, and finally prepared into cell suspension with a density of 1.0×106/mL. Rat SAP models were created by retrograde injection of 3.5% sodium deoxycholate into the biliopancreatic duct. Thirty-six SD rats with successful model induction were randomly divided in four groups, and underwent intraperitoneal injection of normal saline (model group), intraperitoneal injection of MSC suspension (intraperitoneal injection group), tail vein injection of MSC suspension (tail vein injection group), or intraperitoneal injection plus tail vein injection of MSC suspension (combined injection group) at 12 h after modeling, respectively. In addition, another 9 SD rats underwent sham operation (sham operation group), and then received intraperitoneal injection of normal saline of the same volume 12 h after operation. The rats in each group were sacrificed 24 h after injection and the blood and pancreatic tissue samples were harvested. The pathological changes of pancreatic tissue were observed and the degrees of pancreatic damage were scored. The serum levels of amylase (AMS) and lipase (LPS) were measured by iodine colorimetry and methyl halide substrate methods respectively. The contents of IL-6, IL-1β and TNF-α in pancreatic tissue were detected by ELISA assay. The activity of peroxidase (SOD) was detected by hydroxylamine method, and concentration of malondialdehyde (MDA) was detected by thiobarbituric acid method. The mRNA expressions of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), NF-κBp65 were determined by RT-PCR, and the protein expressions of TLR4, MyD88, and NF-κBp65 were examined by Western blot.
Results: The MSCs were spindle-shaped and formed whirlpool-like patterns, with low CD45 expression (1.25%) and high CD90 expression (99.89%) as well as good viability. Compared with sham operation group, in all other groups, the pancreatic tissues showed varying degrees of damage, the serum levels of AMS and LPS, and the pancreatic contents of IL-6, IL-1β, TNF-α and MDA were significantly increased, while the SOD activity was significantly decreased (all P<0.05), and further, the changing amplitudes of all above variables were successively decreased in the order of  model group, intraperitoneal injection group, tail vein injection group and combined injection group, and all differences had a statistical significance (all P<0.05). 
Conclusion: Bone MSC transplantation can inhibit inflammatory response, reduce oxidative stress and repair pancreatic tissue injury in SAP rats. The mechanism may be probably related to the regulation of TLR4/NF-κBp65 pathway.

Key words: Pancreatitis, Acute Necrotizing, Mesenchymal Stem Cell Transplantation, Inflammation, Toll-Like Receptor 4, Rats

中图分类号: 

  • R657.5
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